Deficiency of secreted cryptdin‐4 detected in a mouse model of Crohn fs disease using a new sandwich ELISA

Abstract

Paneth cells in the base of small intestinal crypts secrete α‐defensins in response to bacteria, contribute to innate immunity, and shape the composition of intestinal microbiota. Among mouse α‐defensins, cryptdin‐4 (Crp4) is the most potent microbicide. However, a lack of sensitive assays to quantify α‐defensins released into the intestinal lumen has limited efforts to establish a relationship between secreted α‐defensin and diseases. Here we establish a highly sensitive sandwich ELISA, and analyze secreted Crp4 in feces from a mouse model of Crohn's disease. SD rats were immunized with native Crp4, and monoclonal antibodies recognizing different epitopes were identified and paired to optimize ELISA conditions. To measure levels of secreted Crp4, feces were collected from BALB/c mice and IL10 null, Crohn's disease model mice, and fecal proteins were extracted. The established ELISA was specific and highly sensitive with a Crp4 detection range of 120~1600 pg/ml. The Crp4 concentration measured in feces of normal mice was 20.4 ± 5.1 ng/ml (n=5). In contrast, feces from IL10 null mice contained 8.7 ± 5.0 ng/ml of Crp4 (n=5). Secreted Crp4 was significantly reduced in IL10 null mice. Quantifying secreted α‐defensin in feces enable us to know luminal α‐defensin levels for the first time, and contributes to a deeper understanding of diverse diseases such as inflammatory bowel disease, infectious disease, and obesity.